Computer program for making measurements of accumulations of magnetic particles

ABSTRACT

An apparatus is provided for quantitatively measuring groups of magnetic particles. The particles are complexed with substances to be determined and are excited in a magnetic field. The magnetizations of the magnetic particles are thereby caused to oscillate at the excitation frequency in the manner of a dipole to create their own fields. These fields are inductively coupled to at least one sensor such as sensing coils fabricated in a gradiometer configuration. The output signals from the sensing coils are appropriately amplified and processed to provide useful output indications.

This application is a division of application Ser. No. 09/451,660 filedNov. 30, 1999, which is a continuation-in-part of application Ser. No.08/975,569, Nov. 21, 1997, now U.S. Pat. No. 6,046,585.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to sensing the presence of magneticparticles, and more particularly to quantitatively measuringaccumulations of such particles by means of AC magnetic excitation andinductive sensing of the amplitude of the resulting oscillations of themagnetic moments of the particles at the excitation frequency.

2. Discussion of Prior Art

Much attention has been given to techniques for determining thepresence, and possibly the level of concentration, of minute particlesin a larger mixture or solution in which the particles reside. It isdesirable in certain circumstances to measure very low concentrations ofcertain organic compounds. In medicine, for example, it is very usefulto determine the concentration of a given kind of molecule, usually insolution, which either exists naturally in physiological fluids (forexample, blood or urine) or which has been introduced into the livingsystem (for example, drugs or contaminants).

One broad approach used to detect the presence of a particular compoundof interest, referred to as the analyte, is the immunoassay, in whichdetection of a given molecular species, referred to generally as theligand, is accomplished through the use of a second molecular species,often called the antiligand, or the receptor, which specifically bindsto the first compound of interest. The presence of the ligand ofinterest is detected by measuring, or inferring, either directly orindirectly, the extent of binding of ligand to antiligand.

A discussion of several detection and measurement methods appears inU.S. Pat. No. 4,537,861 (Elings et al.). That patent discloses severalways to accomplish homogenous immunoassays in a solution of a bindingreaction between a ligand and an antiligand, which are typically anantigen and an antibody. Elings discloses creation of a spatial patternformed by a spatial array of separate regions of antiligand material andligand material dispersed to interact with the spatial array of separateregions of antiligand material for producing a binding reaction betweenthe ligand and the antiligand in the spatial patterns and with the boundcomplexes labeled with a particular physical characteristic. After thelabeled bound complexes have been accumulated in the spatial patterns,the equipment is scanned to provide the desired immunoassay. The scannermay be based on fluorescence, optical density, light scattering, colorand reflectance, among others.

The labeled bound complexes are accumulated on specially preparedsurface segments according to Elings, or within an optically transparentconduit or container by applying localized magnetic fields to thesolution where the bound complexes incorporate magnetic carrierparticles. The magnetic particles have a size range of 0.01 to 50microns. Once the bound complexes are accumulated magnetically withinthe solution, the scanning techniques previously described are employed.

Magnetic particles made from magnetite and inert matrix material havelong been used in the field of biochemistry. They range in size from afew nanometers up to a few microns in diameter and may contain from 15%to 100% magnetite. They are often described as superparamagneticparticles or, in the larger size range, as beads. The usual methodologyis to coat the surface of the particles with some biologically activematerial that causes them to bond strongly with specific microscopicobjects or particles of interest (e.g., proteins, viruses, cells, DNAfragments). The particles then become “handles” by which the objects canbe moved or immobilized using a magnetic gradient, usually provided by astrong permanent magnet. Thus, the Elings patent is an example oftagging using magnetic particles. Specially constructed fixtures usingrare-earth magnets and iron pole pieces are commercially available forthis purpose.

Although these magnetic particles have only been used in practice formoving or immobilizing the bound objects, some experimental work hasbeen done on using the particles as tags for detecting the presence ofthe bound object. This tagging is usually done by radioactive,fluorescent, or phosphorescent molecules which are bound to the objectsof interest. A magnetic tag, if detectable in sufficiently smallamounts, would be very attractive because the other tagging techniquesall have various important weaknesses. For example, radioactive methodspresent health and disposal problems. The methods are also relativelyslow. Fluorescent or phosphorescent techniques are limited in theirquantitative accuracy and dynamic range because emitted photons may beabsorbed by other materials in the sample. See Japanese PatentPublication 63-90765, published Apr. 21, 1988 (Fujiwara et al.).

Because the signal from a very tiny volume of magnetic particles isexceedingly small, it has been natural that researchers have triedbuilding detectors based on Superconducting Quantum Interference Devices(“SQUID”s). SQUID amplifiers are well known to be the most sensitivedetectors of magnetic fields in many situations. There are severalsubstantial difficulties with this approach, however. Since the pickuploops of the SQUID must be maintained at cryogenic temperatures, thesample must be cooled to obtain a very close coupling to these loops.This procedure makes the measurements unacceptably tedious. The generalcomplexity of SQUIDs and cryogenic components renders them generallyunsuitable for use in an inexpensive desktop instrument. Even a designbased on so-called “high Tc” superconductors would not completelyovercome these objections, and would introduce several new difficulties.See Fujiwara et al.

There have been more traditional approaches to detecting and quantifyingthe magnetic particles. These have involved some form of forcemagnetometry in which the sample is placed in a strong magnetic gradientand the resulting force on the sample is measured, typically bymonitoring the apparent weight change of the sample as the gradient ischanged. An example of this technique is shown in U.S. Pat. Nos.5,445,970 and 5,445,971 to Rohr. A more sophisticated technique measuresthe effect of the particle on the deflection or vibration of amicromachined cantilever. See Baselt et al., A Biosensor based on ForceMicroscope Technology, Naval Research Lab., J. Vac Science Tec. B., Vol14, No. 2 (pg. 5) (April 1996). These approaches are all limited in thatthey rely on converting an intrinsically magnetic effect into amechanical response. This response must then be distinguished from alarge assortment of other mechanical effects such as vibration,viscosity, and buoyancy.

There would be important applications for an inexpensive,room-temperature, desktop instrument which could directly sense andquantify very small amounts of magnetic particles.

SUMMARY OF THE INVENTION

Broadly speaking, the present invention provides a method and anapparatus for directly sensing and measuring very small accumulations ofmagnetically susceptible particles, e.g., magnetite, and consequently,their coupled substances of interest.

The magnetic particles or beads are coupled by known methods to analyteparticles, thereby providing magnetic sample elements or magnetic boundcomplexes. A well-defined pattern of the magnetic sample elements isdeposited on a surface on a holder. The surface may be flat. Ahigh-amplitude, high-frequency magnetic field is then applied to excitethe particles in the sample. The field causes the particles to behave asa localized dipole oscillating at the excitation frequency. The fieldsfrom the sample are closely coupled to a sensor, such as an array ofinductive sensing coils, which may be fabricated in a gradiometerconfiguration. This configuration makes the sensing coils mostlyinsensitive to the large, uniform field that is used to excite thesample. Moreover, the geometry of the coils is designed to match thespatial pattern of the sample so as to provide a large response thatvaries distinctively with the relative positions of the sample andcoils.

The voltage induced across the sensor is carefully amplified andprocessed by phase-sensitive detection. An inductive pickup from thedrive field itself may serve as the reference signal to the phasedetector circuit. The output of the phase detector is further filteredand digitized.

The signal amplitude is modulated by moving the sample with respect tothe sensor. This allows the rejection of signals due solely to imbalanceof the sensor, non-uniformity of the drive field, cross-talk in thecircuitry, or any other source of apparent signal which is not due tothe sample itself. The digitized shape of the signal amplitude withrespect to the sample position is compared to the theoretical responseshape using appropriate curve-fitting techniques, providing a veryaccurate estimate of the magnetic content of the sample in the face ofinherent instrument noise and drift.

BRIEF DESCRIPTION OF THE DRAWINGS

The object, advantages and features of this invention will be moreclearly seen from the following detailed description, when read inconjunction with the accompanying drawings, in which:

FIG. 1 is a perspective view of a desktop version of an embodiment ofthe present invention;

FIG. 2 is an enlarged plan view of an embodiment of the sensor, showingsensing coils in the embodiment of FIG. 1;

FIG. 3 is a mechanical schematic perspective view of the embodiment ofFIG. 1;

FIG. 4 is an electrical schematic diagram of the embodiment of FIG. 1;

FIG. 4A is an enlarged plan view of the substrate holding the sensingcoils of FIG. 1;

FIG. 4B is a perspective view of a metal shield for the connection endof the substrate;

FIG. 5 is an enlarged plan view of an alternative embodiment of thesensing coils of the embodiment of FIG. 1;

FIG. 6 is a signal waveform of the output of the sensing coils versusthe position of the magnetic material;

FIG. 7 is an embodiment of a lateral flow membrane sample holder whichmay be used in an embodiment of the present invention;

FIG. 8 is an E-core magnet system which may be used as the magneticfield source according to an embodiment of the invention (note that nodrive coils are shown for clarity);

FIG. 9 is an embodiment of a microfluidic sample holder which may beused in an embodiment of the present invention; and

FIG. 10 is an embodiment of a single magnet pole piece with attachedsensor which may be used in an embodiment of the present invention.

DETAILED DESCRIPTION

Referring now to the drawing, and more particularly to FIGS. 1 and 3thereof, there is shown a preferred embodiment of the invention.

I. Reader Module

The reader module includes several distinct subsystems. These include: asample holder with a motion control. The magnetic bound complex samplesfor measurement reside on the holder, and the same also provides thenecessary relative motion within the system. A magnetizer or magneticfield source applies the excitation signals to the samples. Sensors,such as sensing coils, act as the signal pick-up for the signalsgenerated in the samples. A drive circuit supplies the drive current tothe coils of the magnetic field source. An amplifier/phasedetector/digitizer is coupled to the sensor to receive and process theoutput signals therefrom. A microcomputer chip provides two-waycommunication between the external personal computer (PC) and the readermodule.

A. Sample Motion Control

Magnetic particles are coupled to analyte or target particles byconventional methods to create magnetic bound complex samples. Theanalyte particles may include atoms, individual molecules and biologicalcells, among others. It is noted here that the terms “target particle”and “analyte particle” are used substantially interchangeably. It isfurther noted that the term “target” is not intended to be limited tothe definition of that term as used in the field of DNA recombinanttechnology.

The magnetic bound complex samples are deposited in accumulations ofseveral to several hundred particles at a number of predeterminedpositions 11 near the perimeter of a sample holder, such as disc 12(FIG. 3). Other sample holders which may be substituted include lateralflow membranes, plastic strips, or holders employing lateral flow butwithout membranes. An embodiment employing lateral flow membranes isdescribed in more detail below.

Another type of sample holder may employ microfluidics. A microfluidicssystem may have a sample sensing chamber and appropriate channeling tomove a sample in or out of the sensing chamber using variations inpressure. For example, referring to FIG. 9, a microfluidic system 151 isshown having an inlet channel 152. The inlet channel 152 is connected toa mixing chamber 164. A number of reagent chambers 154, 156, and 158 maybe provided to hold various chemicals or reagents. As described below,they may also hold magnetically susceptible particles if desired. Nearthe periphery, or elsewhere, a sample analysis chamber 166 may belocated. The location of this chamber is a predefined location and iswhere the sample magnetic measurement would occur. Accordingly, thesample holder must be configured to allow this chamber to be accessibleto the sensor and the magnetic field source. Otherwise, the magneticmeasurement may proceed as described elsewhere in this specification.Further processing may occur after the magnetic measurement. For thisreason, a measurement chamber 168 is provided, which may also have itsown reagent chamber 160. More reagent chambers may be provided ifdesired. An optional outlet or exit channel 162 may be provided. Suchchannels may not be necessary if the device is only a single-use device.Not shown in this figure for convenience but which may also be providedare various pressure inlets and valves which allow analyte particles,magnetically susceptible particles, and reagents to be shuttled aroundfrom chamber to chamber.

Analyte particles may be quantitatively measured via measuring theirbound magnetically susceptible particles. In the microfluidic system,the samples may be introduced via the inlet channel as combinations ofanalyte and magnetically susceptible particles. Alternatively, theanalyte particles may be introduced via the inlet channel and the twomay be combined and mixed in the mixing chamber 164.

Variations of this system may be manyfold. For example, the sensor maybe located directly on the microfluidic chip to match the region ofanalysis especially well. In another variation, a different parameter onthe chip may be varied at the same time or at a different time, such astemperature. Temperature control means may be located on the chip oroutside of the chip, such as in the case of laser heating within themixing chamber. Such a system requires an optical window, as would beunderstood. Other parameters which may be varied may be anything thataffects the presence or property of the magnetic tag, i.e., themagnetically susceptible particle, or its binding to the analyteparticle.

The ways the bound complexes may be adhered to the pre-defined spots onthe disc are known and may employ standard technology. The disc ismounted on an axial shaft 13 which extends downwardly to a toothed wheel14. An appropriate rotational device, such as a stepper motor 16, has ashaft 17 extending therefrom with a worm gear member 15 at the distalend thereof. The motor provides controlled rotary motion of disc 12pursuant to signals applied from a PC 66 through a number of wires 18.Of course, wireless coupling between the PC and the system of theinvention could be used if desired.

In one preferred embodiment, as presently contemplated, disc 12 is about47 mm in diameter and about 0.25 mm thick. It may be made of glass,plastic or silicon, for example. Its thickness range, for practicalfunctional purposes, would be about 0.1 mm to about 1.0 mm.

In the case where the sample holder is a lateral flow membrane, thesample holder may be made partially porous so that passage of theanalyte particles through the porous portion of the holder may beanother parameter to be varied. In this case, the magneticallysusceptible particles may be bound to the porous sample holder. Forexample, passage of the analyte particles through a porous portion of aholder may likely depend on the mass or size of the particles. Thus, thelocation of the particles within the porous portion may bemass-dependent or size-dependent. As the analyte particles pass throughthe porous sample holder, they may bind preferentially and in apredetermined manner to the bound magnetically susceptible particles.The bound samples, containing analyte particles combined withmagnetically susceptible particles, may then be measured magneticallyusing the device embodied herein. The porous portion of the holder maybe replaced with, e.g., a filter as is known in the art. Such filtersmay be chosen to provide a suitable mass- or size- dependency accordingto the requirements of the process.

For example, referring to FIG. 7, a lateral flow membrane 101 is shown.Analyte particles may be flushed into a release pad 102 where they arereleased into a flow membrane 103. The particles may then flow bycapillary action down the membrane and past a test line 106 on whichbound magnetically susceptible particles are located. A control line 108may also be provided. Finally, an absorbent pad 104 may be locateddownstream if desired to collect the unbound analyte particles.

In operation, the test line may include colloidal iron particles coatedwith a material that specifically binds to a material in the analyte ofinterest. In this way, the test line collects analyte particlespreferentially. The control line 108 may have a known amount ofcolloidal iron for calibration or other such purposes. It should beclear that such a lateral flow membrane may be replaced with, e.g., agel electrophoresis test area. In this case, of course, the samples arenot immobilized but may be moving past the sensing area.

The sample holder may also employ a reference device, such as a barcode, to provide a unique machine-readable tag to identify or locate anindividual region or regions and the assay(s) that are associatedthereby. The reference device may spatially index the location of anindividual region or regions of analysis. The reference device gives aconvenient way to identify a sample of magnetic complex material.Besides bar codes, the reference device may alternatively employ amagnetic strip, a microchip, an optical reference, and so on. Thereference device may be optically aligned with its corresponding samplefor ease of reference.

The computer/CPU may read the reference information along with themagnetic (assay) signal and then display and store the assay results inthe appropriate context. For example, an assay to measure the presenceof e. coli would likely have results displayed in a different form thanan assay testing for the presence of binding of oligonucleotides. Sincethe substrate may be prepared specifically for each kind of assay, thisinformation can be encoded on the substrate as a bar code or using oneof the techniques described above.

In this particular exemplary embodiment, motor 16 rotates wheel 14,which is connected to disc 12 by shaft 13, through a 120-tooth worm gearreduction. Of course, rotational drives having different particularscould also be employed.

A magnetic field source 21 may be moved linearly with respect to disc 12by a rotational device, such as a stepper motor 22, having a 40turn-per-circle lead screw 23 on a motor shaft 24. A boss 25 isconfigured with a hole having internal threads to which the spiral leadscrew threads are coupled. The control signals are applied frommicrocomputer 65 to motor 22 through a number of wires 26. Again, thespecifics of the rotational drive are set out here as an example only.Other appropriate elements having different characteristics could alsobe used.

For example, while the above system describes a situation where themagnetic field source is moved linearly with respect to the sampleholder, another embodiment may be used in which the sample holder ismoved relative to the magnetic field source. In this latter embodiment,the sample holder may be mounted to a shaft and mechanical drive systemsimilar to the drive system shown in FIG. 3. The drive system may movethe sample holder into the gap of the magnetic field source in acontrolled manner.

Numerous types of drive systems may be employed. These include steppermotors, screw and motor arrangements, hydraulics, magnetic drives,configurations in which a human operator physically moves the sampleholder relative to the magnetic field source and relative to the sensor,pressure drives, pinch rollers, conveyor systems, etc.

The above describes the motion of the sample holder from a location inwhich samples may be loaded, such as on a disc, to a location near themagnetic field caused by the magnetic field source. Another motion thatoccurs in the system is the movement of the sample holder past thesensor. Various motions may be caused to accommodate this. For example,two-dimensional motion may be accommodated between the sensor and thesample holder. In the embodiment of FIG. 3, one degree of freedom motion(e.g., along an arc of a circle) is shown using motor 16. The drivesystem of motor 22 may also be employed to translate the sensor alonganother degree of freedom. Alternatively, another motor may be used tomove the sample holder 12 along a similar degree of freedom. Finally, itshould be noted that, by using appropriate gearing, the same motor maybe used to provide any combination of the above or different motions.

In other exemplary embodiments, the drive system may include a pinchroller which grasps a plastic strip on which a sample is disposed,moving the same past the sensor in a controlled fashion. Such anembodiment may be particularly useful where the sample is placed in astrip on a plastic card similar to a credit card, which is then“grabbed” by a device similar to that used in ATM machines. Of course,the drive system may also be any of the systems described above as wellas other alternate systems.

B. Magnetic Field Source

Referring to FIG. 4, a ferrite toroid core 31, which is about 30 mm indiameter in the particular embodiment being described, is formed with agap 32, which is about 1.5 mm wide. A drive coil 33 is wound as a singlelayer over about 270 of toroid 31, symmetric with respect to the gap. Afeedback loop 34 encircles the toroid body at a location about 180 from(opposite) the gap. Loop 34 may be outside of coil 33 or between coil 33and the toroid core. It may include a few or many turns, as necessaryand appropriate for the feedback function. The purpose of the feedbackloop is to sense or represent the field in gap 32 and enable the signalprocessing or output circuit to self-correct for variations such astemperature drift. This loop is used to enhance precision and is notessential to proper operation of the system.

Various other magnetic field sources may also be used. For example,while most all employ electromagnets, the electromagnets may be in theform of, e.g., toroids or so-called “E-core”s which are magnetsemploying the shape of an “E” (see FIG. 8). In E-cores, the middlesegment of the “E” is made somewhat shorter than the outer segments.Referring to FIG. 8, two E-cores 112 and 112′ are placed with their opensides facing each other. The shorter middle segments then define a smallgap 114 therebetween. A sample on, e.g., a plastic strip 116 may then besituated in this small gap. The sensor used to measure the oscillationof the magnetizations may be on a separate substrate 118 also located inthe small gap or may alternatively be disposed on the end of one or bothof the shorter middle segments. In any of the embodiments, in fact, thesensor may be disposed on a magnetic pole piece or other such elementthat forms a perimeter of the gap. In this way, the unit may be mademore modular and the coil placement more uniform and consistent.

In other embodiments, no gap is needed at all. Referring to FIG. 10, asingle magnetic pole piece 201 may be situated with a sensor disposedthereon or disposed on a separate strip. In FIG. 10, the sensor is shownas two sensing coils 202 and 204. The pole piece can alternate themagnetic field, and the sensor can measure the oscillatingmagnetizations as above.

Referring back to FIG. 3, the toroidal magnetic field source assembly ismounted in insulative housing 35, which may be formed from fiberglass.Housing 35 has a slot 36 corresponding to the position of gap 32. Thisslot/gap is shaped and configured to selectively receive the edge ofrotatable disc 12, and provides space for the sensing coil substrate,which is described in detail below.

C. Sensors

A sensor is used to measure the magnetic field strength of the samples.In this embodiment, the method used is AC susceptibility. A number oftypes of sensors may be employed. In the embodiments below, sensingcoils connected in a gradiometer configuration are described. It shouldbe noted that the gradiometer configuration is not necessarily required;moreover, other types of sensors may be used. These sensors may includeHall sensors, GMR sensors, or other such sensors capable of measuringmagnetic field strength or magnetic flux.

With particular reference now to FIGS. 2, 4 and 4A, insulative substrate41 is disposed in slot 36 in housing 35 and extends into gap 32. Bondingpads 40, 42 are provided at a proximal end of substrate 41 and a sensor,in particular sensing coils 43, is mounted adjacent a distal end ofsubstrate 41. Preferably the substrate is made of sapphire or siliconand the sensing elements are thin film copper coils. Standard thin filmfabrication techniques can be used to construct the substrate andsensing coils, where the leads to and from each coil are on separatedifferent layers. For example, incoming traces 49 may be laid on thesubstrate surface by standard photolithographic processing methods, alayer of sputtered quartz may then cover the incoming leads, then coils43 and output leads 44 are similarly applied and a protective layer ofquartz may then be added on top. The usual means for connecting betweenthe layers would be used.

The sensing coils, which are connected in series opposition creating agradiometer configuration, are connected to bonding pads 40 and 42 byconductive traces 44 and 49, and thence to signal processing circuitryby twisted-pair wires 45. The twisted pair arrangement is employed toassist in reducing stray signal or interference pickup.

In the spiral form shown in FIG. 2, the coil traces may be about 5microns in width with about a 10-micron pitch between spiral traces. Thethickness of the sensing coil traces may be about 1 micron. The diameterof each completed coil is about 0.25 mm.

By making substrate 41 relatively long and narrow, bonding pads 40, 42are relatively far away from the toroid gap, which helps minimize straypickup in soldered leads 45. Metal shield 46 (FIG. 4B) may be employedaround the bonding area to further contribute to the reduction of straysignals or interference pickup. The shield is essentially a short pieceof a thick-walled cylinder, typically formed of copper. The shieldprovides electrical shielding and facilitates mechanical handling, butis not essential to operation of the embodiment of the invention. Theconnection (proximal) end of the substrate is slid into slot 50 afterthe wire connections are made.

An alternative embodiment of the sensing coils is shown in FIG. 5. Theplanar configuration of coils 47 is an elongated rectangle. The tracedimensions are about the same as for the FIG. 2 coils and the compositecoil width is also about 0.25 mm. The coil length is about 1-2 mm andthe coils are connected to bonding pads 52, 53 by means of leads 48, 51.

In another-alternative embodiment, two sets of coils may be used. Oneset of coils may be used as described above, to measure the magneticmoment of the sample. Another set of coils may be employed within thesame substrate as a reference set of coils. This reference set of coilsmay be disposed, e.g., on the side of the substrate opposite that of thesample set of coils. In any case, the reference set of coils is disposedfar enough from the sample that the effect of the sample magnetic momentis not detected by the reference set of coils. The reference set ofcoils is then used to measure the strength of the signal from ananalysis region containing a predetermined amount of magnetic materialor reference analyte. By comparison of the magnetic field detected bythe sample set of coils with the magnetic field detected by thereference set of coils, an even more accurate measurement of the samplemagnetic moment may be made. To provide another reference, a magneticstandard may be employed as one of the samples. When such a standardsample is measured, the results may be used to calibrate the system forfuture or previous measurements. This calibration may significantly helpto reduce noise in the system. Auto-calibration may also be employedwith such a system, using the differential between signals, to zero thesignal.

D. Drive Circuit

The magnetic drive circuit, shown at the left side of FIG. 4, is builtaround a pair of high-current, high-speed operational amplifiers 54, 55.With the power provided by transformer primary winding 56, theamplifiers can provide in excess of about one ampere of drive current tomagnetizing or drive coil 33 at about 200 kHz. This drive circuit ishighly balanced to minimize common-mode noise pickup in sensing loops orcoils 43, 47.

Small secondary winding 57 coupled to loop 34 around the magnetizingcoil provides a feedback voltage to operational amplifiers 54 and 55 tosustain oscillations at a well-regulated amplitude and frequency. Thissecondary winding 57 also provides an optimum reference signal for thephase-detector circuitry, described below.

This embodiment describes an alternating field as the driving source forthe complex of magnetic and analyte particles. In a separate embodiment,the driving source may be non-sinusoidal, e.g., may be a field pulse ora square wave. A variety of other such waveforms may also be used.

E. Amplifier/Phase Detector/Digitizer

A low-noise integrated instrumentation amplifier is the basis for thiscircuitry, although somewhat better noise performance could be obtainedusing discrete components. Amplifier 61 is transformer coupled to thesensing coils in order to reduce common-mode noise signals and tofacilitate a convenient way to null out imbalance in the magnetic fieldsource and in the sensor. The transformer coupling is conventional, islocated in amplifier 61, and is not specifically shown in the drawing.In an alternative embodiment, amplifier 61 may be replaced by orsupplemented with a preamplifier disposed on the substrate. In otherwords, substrate 41 may have patterned thereon a preamplifier to modifythe signals from the sensor prior to the phase-sensitive detection step.Phase sensitive detector 62 is also designed around a special purposeintegrated circuit. Phase sensitive detector 62 may be a phase-lockingdevice or alternatively any other type of phase-sensitive device. Theoutput of the phase detector is applied to low-pass filter 63 and isthen digitized in A/D converter 64. The converter may be a highresolution, 20-bit sigma-delta converter, for example. Such a converterchip has adequate hum rejection at both 60 and 50 Hz, which proves to bevery helpful in maximizing the sensitivity of the instrument. It is anoff-the-shelf item, available from several manufacturers.

F. Microcomputer

Microcomputer 65 includes a microprocessor chip, such as a MotorolaHC11, and has a built-in port which supports two-way serialcommunication to PC 66 by plugging into the serial port of the PC. Italso has specialized ports for communication with serial A/D converter64 and stepper motors 16 and 22. A simple command language programmeddirectly into microcomputer 65 allows the PC to send commands andreceive responses and data.

Microcomputer 65 may also perform many of the functions previouslydescribed above. For example, microcomputer 65 may be equipped with aphase-sensitive device of its own, such as a digital lock-in. Such amicrocomputer 65 may acquire the signals, separate data from noise, anddisplay the results.

G. Human Interface

The PC provides the operational command for the system. The PC runs thesystem through an RS-232 interface, e.g., from the microcomputer. The PCprovides a display of the results of the measurements. The display maybe, e.g., a computer monitor display or any other form ofcomputer-assisted readout.

II. Operation of the System

In a relatively straightforward and known manner, a well-defined dot orpattern of the magnetic particle complexes comprising the samples isdeposited on disc 12 at one or more locations 11 near the peripherythereof. Pursuant to control signals from the PC, stepper motor 22 isenergized to rotate lead screw 23 to move the magnetic field sourceassembly towards sample disc 12. When a sample position 11 near theperipheral edge of disc 12 is aligned with a sensor such as sensingcoils 43, 47 in the middle of toroidal gap 32, stepper motor 22 stopsand a high amplitude (1 ampere, for example), high frequency (200 kHz)signal is applied to toroidal drive coil 33. Again, while sensing coilsare described below, it should be understood that a variety of sensorsmay be employed. A signal from PC 66 then energizes stepper motor 16 torotate the disc and thereby move the sample dot past the sensing coils.The high amplitude, high frequency magnetic field in gap 32 therebyexcites the magnetic particles of the sample in the gap. The appliedcurrent is intended to drive the toroid to saturation, resulting in thefield in the gap have a magnitude of about 1000 oersted. The particlesthen oscillate magnetically at the excitation frequency, behaving as alocalized dipole. Given the close physical proximity of the magneticparticles to the sensing coils, the magnetic fields from the sample areclosely coupled to the gradiometer configured sensing coils. Because ofthe gradiometer configuration of the sensing coils, the output of thesensing coils due to the large, uniform excitation field issubstantially null or zero. In order to obtain the largest possibleresponse, the geometry of the sensing coils is configured to match thespatial pattern of the samples. That is, the sample pattern dots are nolarger than about 0.25 mm across. The response signal variesdistinctively with the relative position of the sample and the coils.

The signal from the sensing coils in the presence of the drive field andin the absence of a sample may serve as the reference signal to thesignal processing portion of the system. As the sample moves past onesensing coil and then the other, the phase of the coil output signalreverses by 180 as shown in FIG. 6, thereby providing a very powerfuldetection technique. As shown in FIG. 6, the output may be shown as theresponse of the sensing coils versus the position of the sample withrespect to the sensing coils. The induced voltage is amplified byamplifier 61 and processed by phase detector 62. That signal is filteredand digitized and passed to the PC through microcomputer 65 to providethe output signals to the PC. Indicator 67 may be any type of useabledevice to provide information to the system operator. Indicator 67 couldbe a visual indicator, conveying information numerically or graphically,or could also be a variety of lighting systems, audible indicators, orany combination of these or other possible indicators.

The output signal amplitude is modulated by moving the sample withrespect to the array of the sensing coils. This permits rejection ofsignals due solely to system and external inputs and not due to thesample itself. The digitized shape of the signal amplitude with respectto sample position is compared to the theoretical response shape storedin PC 66 using appropriate curve fitting techniques. These techniquesmay include phase-sensitive techniques or other techniques yieldingsimilar results. The result of this operation is a very accurateestimate of the magnetic content of the sample to the exclusion ofinherent instrument noise and drift.

While a preferred embodiment of the invention has been presented above,some alternatives should be mentioned. Two sensor coil shapes have beenshown but numerous other configurations may be employed. Moreover, asindicated above, sensors may be used which are patterned directly on oneor more of the magnetic field source pole pieces. Furthermore, othervarieties of sensors could be employed besides the types of coilsdisclosed. For example, balance hall sensors may be employed. Inappropriate configurations, these may yield a frequency independentsignal. Other sensors which may be advantageously employed include giantmagnetoresistance (GMR) sensors, SQUID sensors, magneto-resistancesensors, etc.

In other variations, the magnetic field source is shown as moving withrespect to the sample disc, but the disc and coupled stepper motor couldbe configured to move with respect to the magnetic drive assembly ifdesired. The toroid core is shown with a rectangular cross section butother shapes are also feasible. As to the number of sample particles ina dot 11 on disc 12, by way of example, a 0.25 mm dot of sample elementscould contain about 10 five-micron size magnetic particles, or about1200 one-micron size particles.

Thus, in view of the above description, it is possible thatmodifications and improvements may occur to those skilled in theapplicable technical field which are within the spirit and scope of theaccompanying claims.

What is claimed is:
 1. A computer program, residing on acomputer-readable medium, for quantitatively measuring analyte particlescombined with magnetically susceptible particles which form boundcomplex samples, the computer program comprising instructions forcausing an apparatus to: create a magnetic field; excite themagnetically susceptible particles, that are bound with analyteparticles and which form bound complex samples, and cause oscillationsof the magnetizations therein; sense the fields generated by theoscillating magnetizations; and create a signal representative of thesensed fields.